Bovine cells expressing adenovirus essential functions for propagation of recombinant adenoviral vectors

ABSTRACT

The invention provides cell lines capable of supporting the replication of a defective recombinant virus vector. In one aspect, bovine cell lines expressing adenovirus E 1  functions are provided. The cell lines are useful for the propagation of adenovirus vectors with mutations and/or deletions in E 1  and other essential regions of the adenovirus genome.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patentapplication Serial No. 60/155,219, filed on Nov. 2, 1998.

TECHNICAL FIELD

[0002] The invention is in the fields of recombinant cell lines,recombinant animal viral vectors, defective adenovirus vectors, subunitvaccines and gene therapy.

BACKGROUND

[0003] Adenoviruses have recently begun to be used as vectors for geneexpression, recombinant subunit vaccines and gene therapy. Yeh el al.(1997) FASEB J. 11:615-623; and Imler (1998) Vaccine 13:1143-1151. Theyhave been detected in many animal species, exhibit minimalpathogenicity, and are non-integrative. Adenoviruses are capable ofinfecting a wide variety of cell types, both dividing and quiescent, andhave a natural tropism for airway epithelial cells. The advantages tothe use of adenoviruses as vectors include suitability for geneticmanipulation, ability to replicate to high titers, stability and ease ofproduction. Adenoviruses have been used as live enteric viral vaccinesfor many years with an excellent safety profile.

[0004] Adenoviruses are distinguished according to the species of animalwhich they infect (e.g., human, bovine, canine, etc.). Particularspecies of adenoviruses are further characterized serologically,according to type.

[0005] The adenovirus E1 region encodes several functions that areessential to viral replication. The E1A region is responsible forencoding functions that activate early and late transcription, stimulateprogression of infected cells into the S phase of the cell cycle, andantagonize the effects of α- and β-interferons. The E1B region encodesfunctions involved in stimulating cell-cycle progression of infectedcells, blocking apoptosis in infected cells, and blockingnuclcocytoplasmic transport of host cell mRNA. In addition, part or allof the E1 region is responsible for cell transformation. See, forexample, Shenk, Adenoviridae: The viruses and their replication. In“Virology” (B. Fields, ed.) Chapter 67, Lippincott-Raven, Philadelphia,1996, pp. 2111-2148.

[0006] Ideally (for safety considerations), one or more essentialregions of the adenovirus genome are inactivated in the genome of anadenoviral vector. For example, the E1 region, encoding severalessential functions (see above) as well as potential adenovirustransforming functions, will be inactivated in many types of adenovirusvector. However, since the E1 regions are essential for normal virusreplication, propagation of adenovirus vectors lacking all or part ofthe E1 regions requires a helper cell line that provides E1 functions.Heretofore, such helper cell lines have provided E1 function byincluding E1 sequences from the same adenovirus type that is propagatedin the cell line. For example, the human 293 cell line, containing humanadenovirus type 5 E1 sequences, can be used for the propagation of humanadenovirus with a mutated E1 region. Graham et al. (1977) J Gen. Virol.36:59-72. Similarly, cell lines suitable for the propagation of E2- andE4-mutant adenoviruses have been described. Klessig et al. (1984) Mol.Cell. Biol. 4:1354-1362; Weinberg et al. (1983) Proc. Natl. Acad. Sci.USA 80:5383-5386.

[0007] Homologous recombination occurs readily between adenoviruses ofthe same type, both in the wild and during coinfection of culturedcells. Ginsberg et al The Genetics of Adenoviruses. In: Fraenkel-ConratH and Wagner RR eds., “Comprehensive Virology” volume 9, New York,Plenum Press, 1977; Takemori (1972) Virology 47:157-167; and Williams etal. (1975) Cell 4:113-119.Consequently, when a mutant adenovirus ispassaged through a helper cell line containing homologous adenovirussequences, homologous recombination can result in the generation ofwild-type adenoviruses. For example, when replication-defectiveadenoviruses containing E1 deletions were passaged in a complementingcell line containing adenovirus E1 sequences, replication-competentviruses emerged, in which the deleted E1 region had been restoredthrough recombination with homologous E1 sequences present in the helpercell. See, for example, Hehir et a. (1996) J Virol. 70:8459-8467;Fallaux et al. (1998) Human Gene Therapy 9:1909-1917.

[0008] Accordingly, there is a need for an adenovirus vector-helper cellsystem in which vectors deleted for E1 can be propagated in a cell lineproviding E1 function, without the likelihood that wild-type virus willbe generated by recombination between the vector genome and viralsequences in the helper cell.

SUMMARY OF THE INVENTION

[0009] It is an object of the invention to provide a system for thegrowth and propagation of replication-defective adenovirus vectors,wherein the system does not have the potential to produce recombinant,replication-competent adenoviruses.

[0010] Accordingly, the invention provides host cells, preferablybovine, that are permissive for the replication of a defectiveadenovirus vector, in particular, a recombinant adenovirus that ismutated in the E1 region of the adenovirus genome (i.e., the E1A region,the E1B region or both). The E1 mutation can be a deletion, insertion,substitution, one or more point mutation(s), a rearrangement, or anyother type of in vivo or in vitro genetic change. Such defectiveadenovirus vectors will often comprise heterologous sequences. Inadenovirus genomes with deletions in E1, the heterologous sequences canbe inserted at or near the site formerly occupied by the deleted E1sequences, and/or at any other region of the genome. Adenovirus genomesthat are mutant in their E1 region can also contain mutations in otherregions of the genome, such as the E3 region or the region between E4and the right end of the genome.

[0011] In one embodiment, host cells comprise E1 sequences from anadenovirus of a different type or a different species than theadenovirus vector that is propagated in the host cells. In a preferredembodiment, the host cells comprise human adenovirus E1 sequences andthe vector that is propagated in the host cells is a bovine adenovirus.

[0012] In one embodiment, the bovine host cells are derived from fetalbovine retina. In a preferred embodiment, fetal bovine retina cellscomprise adenovirus E1 sequences that have been introduced into thecells by transfection. In a more preferred embodiment, the E1 sequencesare derived from a human adenovirus, for example, human adenovirus type5 (HAd-5). In a still more preferred embodiment, fetal bovine retinacells, comprising HAd-5 sequences are used for the propagation ofreplication-defective bovine adenovirus (BAV) vectors having one or moremutations in their E1 region and, optionally, one or more mutations inother regions of their genome. In an even more preferred embodiment, thereplication-defective BAV vectors comprise heterologous sequences,wherein the heterologous sequences can be located in the E1 region ofthe genome of the defective BAV vector and/or at other regions of thegenome.

[0013] The invention provides host cells as described above, as well ashost cells comprising defective BAV vectors mutated in their E1 region,wherein the defective BAV vectors optionally comprise insertedheterologous sequences.

[0014] In addition, the invention provides methods for the propagationof replication-defective recombinant BAV vectors using the host cells ofthe invention, as well as vectors and vector genomes that have beenpropagated using the host cells of the invention. Defective recombinantBAV vectors and their genomes, produced using the methods andcompositions of the invention, are useful as immunogenic compositions.Such immunogenic compositions can be used both prophylactically andtherapeutically. Prophylactically, the immunogenic compositions are usedfor purposes of vaccination to elicit a protective immune response. Intheir therapeutic uses, the immunogenic compositions are used to induceor boost an immune response to an infection, thereby preventing orameliorating the symptoms of disease.

[0015] In addition, defective recombinant BAV vectors and their genomes,produced using the methods and compositions of the invention, are usefulfor the introduction of hcterologous genes into recipient mammaliancells. When such heterologous genes are in operative linkage withappropriate transcriptional regulatory elements, expression of theheterologous gene in the recipient cell is accomplished. Such expressionis useful in the provision of therapeutic gene(s) and/or gene product(s)and thus will play a role in certain aspects of in vitro, in vivo and exvivo genetic intervention in the treatment of disease, and in genetherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows a map of plasmid TG4671. Sequences from the humanAdenovirus type 5 E1 region are indicated by the thick black line.

[0017]FIG. 2 contains micrographs showing cellular morphology. FIG. 2Ashows fetal bovine retina cells (FBRC). FIG. 2B shows VIDO-R2 cells(FBRC transformed by pTG4671).

[0018]FIG. 3 shows analysis of E1A expression in FBRC and VIDO-R2 cellsby protein immunoblot analysis. Human 293 cells, which express humanadenovirus type 5 E1A and E1B, are used as a positive control. Lane 1:molecular weight markers; lane 2: 293 cells; lane 3: FBRC; lane 4:VIDO-R2 cells.

[0019]FIG. 4 shows analysis of E1B expression in FBRC and VIDO-R2 cellsby protein immunoblot analysis. Human 293 cells, which express humanadenovirus type 5 E1A and E1 B, are used as a positive control. Lane 1:molecular weight markers; lane 2: 293 cells; lane 3: FBRC; lane 4:VIDO-R2 cells.

[0020]FIG. 5 shows a map of plasmid TG5435, comprising a BAV genome. BAVgenes are designated by the thick arrows inside the circle.

[0021]FIG. 6 is a schematic diagram showing the construction ofBAV3.500.See Example 4.

[0022]FIG. 7 is a schematic diagram showing the construction ofBAV3.501. See Example 5.

[0023]FIG. 8 is a schematic diagram showing the construction ofBAV3.502. See Example 6.

[0024]FIG. 9 is a schematic diagram showing the construction ofBAV3.304. See Example 7.

[0025]FIG. 10 shows titers of wild-type and recombinant BAVs afterinfection of FBRC and VIDO R2 cells. See Example 8.

[0026]FIG. 11 shows expression of BHV gD in BAV3.501-infected cells. SeeExample 9. ³⁵S labeled proteins from cell lysates wereimmunoprecipitated with anti-gD monoclonal antibodies and separatedunder reducing conditions on a 10% polyacrylamide-SDS gel. FIG. 11Ashows results in VIDO R2 cells; FIG. 11B shows results in MDBK cells.Lane 1: mock-infected; Lane 2: BAV3-infected; Lane 3: BHV-1-infected;Lanes 4-6: BAV3.501-infected and harvested at 12 hours (lane 4), 24hours (lane 5) and 36 hours (lane 6) after infection. Molecular weightmarkers, in kDa, are indicated at the left side of the figure.

[0027]FIG. 12 shows expression of BCV HE in BAV3.502-infected VIDO R2cells. See Example 10. ³⁵S labeled proteins from cell lysates wereimmunoprecipitated with polyclonal anti-BCV serum and separated underreducing conditions on a 10% polyacrylamide-SDS gel. Lane 1:mock-infected; Lane 2: BAV3-infected; Lane 3: BCV-infected; Lanes 4-6:BAV3.502-infected and harvested at 12 hours (lane 4), 24 hours (lane 5)and 36 hours (lane 6) after infection. Molecular weight markers, in kDa,are indicated at the left side of the figure. Two differentautoradiographic exposures of the gel are shown.

[0028]FIG. 13 shows expression of GFP in BAV3.304-infected MDBK cells.Infected cell lysates were separated by gel electrophoresis, and thegels were blotted and probed with anti-GFP serum. Lanes 1-3:BAV3.304-infected cells harvested as 12 (lane 1), 24 (lane 2) and 36(lane 3) hours after infection; Lane 4: mock-infected; Lane 5: wild-typeBAV-3-infected.

DETAILED DESCRIPTION

[0029] A. General Methods

[0030] The practice of the present invention employs, unless otherwiseindicated, conventional techniques of microbiology, immunology,virology, molecular biology, and recombinant DNA which are within theskill of the art. These techniques are fully explained in theliterature. See, e.g., Maniatis et al., Molecular Cloning. A LaboratoryManual (1982); DNA Cloning. A Practical Approach, vols. I & II (D.Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed. (1984)); NucleicAcid Hybridization (B. Hames & S. Higgins, eds. (1985)); Transcriptionand Translation (B. Hames & S. Higgins, eds. (1984)); Animal CellCulture (R. Freshney, ed. (1986)); Perbal, A Practical Guide toMolecular Cloning (1984); Ausubel, et al., Current Protocols InMolecular Biology, John Wiley & Sons (1987, and annual updates; andSambrook et al., Molecular Cloning: A Laboratory Manual (2^(nd)Edition); vols. I, II & III (1989).

[0031] B. Definitions

[0032] Operably linked or operatively linked refers to a juxtapositionof components (usually sequence elements) wherein the components areconfigured so as to perform their usual function. Thus, for example, acontrol sequence operably linked to a coding sequence is capable ofaffecting the expression of the coding sequence. The components need notbe contiguous to one another so long as the control sequence is capableof exerting its normal regulatory function on the coding sequence.

[0033] Transformation or transfection refers to the process by whichexogenous nucleic acid is introduced into a cell. Methods forintroduction of nucleic acids into cells are well-known to those ofskill in the art and include, for instance, microinjection,electroporation, CaPO₄ co-precipitation, DEAE-dextran-mediated transfer,lipid-mediated transfer, particle bombardment, etc.

[0034] Heterologous sequences refer to non-BAV sequences inserted into arecombinant BAV genome. In some cases, heterologous sequences will besequences encoding all or part of a protein or polypeptide. In somecases, heterologous sequences will comprise a gene of interest or afragment thereof.

[0035] The terms host cell and helper cell are used interchangeably todenote a cell or clone of cells capable of supporting the replication ofan otherwise replication-defective adenovirus vector. Host cellsgenerally provide an essential viral function for which the adenovirusvector is deficient. The term cell line can be used to refer to a cloneof host cells.

[0036] C. Host Cells and Cell lines

[0037] The invention includes a cell or cell line which provides anessential viral function, such that a viral vector lacking that functioncan be propagated in the cell or cell line. In one embodiment, the viralvector is an adenovirus vector and the essential viral function providedby the cell line is an adenoviral function. Thus, in one embodiment, acell or cell line is capable of producing at least some of the proteinsrequired for replication of a defective adenoviral vector which thevector itself cannot produce. The protein provided by the cell or cellline can also be a structural protein, required for maturation and/orassembly of the viral particle. The protein can be involved inreplication, transcription, regulation of these processes or any otheressential viral function. The viral function provided by the cell linedoes not necessarily have to encode a protein, it could also encode anessential RNA.

[0038] The essential function, in one embodiment, is encoded by afragment of an adenovirus genome, which can be modified by mutation,such as deletion and/or addition of nucleotides, point mutation,translocation, inversion, or rearrangement, as long as the mutation doesnot impair the capacity of the adenoviral genome fragment to complementa deficiency in a defective adenoviral vector. The adenoviral genomefragment can be present in the cell of the invention in a plasmid orviral vector or, preferably, can be integrated into the genome of thecell. Methods for introducing a fragment of an adenoviral genome into avector, vectors suitable for such purposes, methods for introducing avector or a nucleic acid fragment into a cell, and methods for directingintegration of a nucleic acid fragment into a cellular genome areconventional techniques that are well-known to those of skill in theart. Thus, a stable helper cell line, expressing adenovirus functionsencoded by adenovirus nucleic acid fragment(s) can be established. Inthe construction of such cell lines, co-transfection (of an adenoviralgenome fragment or a vector containing an adenoviral genome fragment)with a selectable marker (such as a gene conferring antibioticresistance) can be used to aid in detection of transfected cells. Insome cases, the helper cell line can be established without the use of aseparate selectable marker, based on the transforming capabilities ofthe products expressed by the adenoviral genome fragment. See, forexample, FIG. 2.

[0039] In a preferred embodiment, the adenoviral function is E1 functionand the adenoviral vector comprises a defective E1 region. The defectcan be a point mutation, substitution, deletion, insertion, sequencerearrangement or any other type of genetic modification resulting inloss of function. Preferably, the defect is a deletion in the E1 region.

[0040] Thus, in one embodiment, the cells and cell lines of theinvention are capable of providing E1 A, E1B or both E1A and E1Bfunctions of an adenovirus. In a preferred embodiment, the cells andcell lines of the invention comprise adenoviral sequences encoding theaforementioned functions. More preferably, the cells and cell lines ofthe invention express E1A and E1B functions encoded by a humanadenovirus, such as, for example, human adenovirus type 5 (HAd-5). Inthese embodiments, human adenovirus E1 sequences can extend from theinitiation (ATG) codon of the most upstream E1A-encoded polypeptidethrough the stop codon of the most downstream E1 B-encoded polypeptide.However, the E1 sequences can also comprise additional adenoviralsequences at either of the 5′ or 3′ extremities, or both. In a preferredembodiment, the cells and cell lines of the invention comprise E1sequences extending from nucleotides 505-4034 of the HAd-S genome. Inanother embodiment, the cells and cell lines of the invention comprise E1 sequences extending from nucleotides 505-3510 of the HAd-5 genome. Thecomplete sequence of the HAd-5 genome is known to those of skill in theart. Chroboczek et al. (1992) Virology 186:280-285.

[0041] Adenoviral sequences, present in a cell of the invention, can beplaced in operative linkage with suitable control elements, bothtranscriptional and/or translational. Control elements can include thosenormally associated with the adenoviral sequences, or heterologoussequences. For example, adenoviral sequences present in a cell of theinvention can retain the E1A promoter sequences and the E1Bpolyadenylation signal. In another embodiment, the adenoviral sequencesare placed under the control of a suitable heterologous promoter whichis functional in a helper cell line of the invention. The use of aheterologous polyadenylation site is also contemplated. Heterologousregulatory elements can be isolated from any eukaryotic or viral genome.Transcriptional regulatory elements, such as promoters, can beconstitutive or regulatable. A regulatable control element can be eitherpositively or negatively regulated, or both.

[0042] In one embodiment of the invention, recombinant cell lines areproduced by constructing an expression cassette comprising an adenoviralE1 region and transforming host cells therewith to provide complementingcell lines or cultures expressing E1 function. These recombinantcomplementing cell lines are capable of allowing a defective recombinantadenovirus with deleted E1 sequences to replicate and express a desiredforeign gene or fragment thereof which is optionally encoded within therecombinant adenovirus. The replication of defective recombinantadenoviruses with deleted E1 sequences and inserted heterologoussequences in a cell or cell line of the invention results in theproduction of infectious virions capable of expressing the heterologoussequence.

[0043] Recombinant complementing cell lines according to the inventionare capable of allowing a defective recombinant BAV, having a deleted E1gene region, wherein the deleted sequences are optionally replaced byheterologous nucleotide sequences, to replicate and express one or moreforeign genes or fragments thereof encoded by the heterologousnucleotide sequences. BAV vectors with E1 deletions, whereinheterologous sequences are inserted in regions other than E1, can alsobe propagated in these complementing cell lines, and will express theheterologous sequences if they are inserted downstream of a BAV promoteror are inserted in operative linkage with a eukaryotic regulatorysequence. For example, cells and cell lines of the invention are usefulin generating recombinant adenoviruses additionally comprising an E3gene deletion, with the heterologous nucleotide sequence encoding aforeign gene or fragment thereof inserted in place of the deleted E3region.

[0044] Preferred helper cell lines include VIDO-R2 cells, as describedin Example 1, infra. Briefly, the VIDO R2 cell line is a fetal bovineretinal cell line that has been transfected with DNA from the humanadenovirus type 5 (HAd-5) E1 region, and which supports the growth ofE1-deleted BAV vectors and E1-deleted human adenoviruses. The presentinvention shows that the human adenovirus E1 polypeptides produced byVIDO-R2 cells are capable of complementing bovine adenoviruses deficientin E1 function. However, the risk of generating replication-competent,recombinant virus is reduced due to the differences in nucleotidesequence between the HAd and BAV E1 regions. Therefore, E1-deleted BAVvectors can be grown in VIDO-R2 cells without the risk of generatingwild-type BAV by recombination.

[0045] More generally, defective recombinant BAV vectors, lacking one ormore essential functions, can be propagated in appropriate complementingcell lines, wherein a particular complementing cell line provides afunction or functions that is (are) lacking in a particular defectiverecombinant BAV vector. Complementing cell lines can provide viralfunctions through, for example, co-infection with a helper virus whichexpresses the function that the vector lacks, or by integrating orotherwise maintaining in stable form a fragment of a viral genomeencoding a particular viral function.

[0046] The invention also includes a BAV vector that has beenconstructed using the host cells of the invention, and expressionsystems comprising said BAV vectors. In certain embodiments, a BAVvector constructed using the host cells of the invention will compriseone or more heterologous nucleotide sequences. That is, non-BAVsequences can replace part or all of the E3 region, part or all of theE1 region, part or all of the E2 region, part or all of the E4 region,part or all of the region between E4 and the right end of the genome,part or all of the late regions (L1-L7) and/or part or all of theregions occupied by the 33 kD, 52 kD, 100 kD, DBP pol, pTP and pentongenes, and genes IIIA, pV, pVI, pVII, pVIII and pX. Any of theaforementioned regions of the genome can be mutated or deleted, alongwith or instead of the E1 region. The expression system can be usedwherein the heterologous nucleotide sequences are optionally under thecontrol of any other heterologous promoter. BAV vectors can alsocomprise inverted terminal repeat (ITR) sequences and packagingsequences.

[0047] The BAV 33 kD, 52 kD, 100 kD, DBP, pTP, penton (III), pIIIA,pIVa2, pV, pVI, pVII, pVIII and pX genes are essential for viralreplication. BAV vectors comprising deletions in any of these genes, orwhich lack functions encoded by any of these genes, can be used in thepractice of the invention. However, such vectors must be grown in anappropriate complementing host cell (i.e., a helper cell line) providingthe essential viral function(s) missing in the vector. In humanadenoviruses, certain open reading frames in the E4 region (ORF 3 andORF 6/7) are essential for viral replication. Deletions in analogousopen reading frames in the E4 region of BAV-3 could necessitate the useof a helper cell line for growth of the viral vector. Accordingly, hostcells providing any of the functions encoded by the genes describedabove are useful in the practice of the invention. Preferred host celllines comprise sequences encoding the human adenovirus counterparts ofthese BAV genes.

[0048] The cell lines and host cells of the invention can be derivedfrom any tissue of any mammalian species. A preferred species of cell isbovine cells. Preferred among bovine cells are those from kidney andfetal retina.

[0049] D. Adenoviral Vectors

[0050] In one embodiment of the invention, a recombinant expressioncassette can be introduced within a BAV vector by cleaving a wild-typeBAV genome with one or more appropriate restriction enzyme(s) to producea BAV restriction fragment comprising E1 or E3 region sequences,respectively. The BAV restriction fragment can be inserted into acloning vehicle, such as a plasmid, and thereafter at least oneheterologous sequence (which may or may not encode a foreign protein),optionally in operative linkage with eukaryotic transcriptional and/ortranslational regulatory sequences, can be inserted into the E1 or E3region. The resulting plasmid or linearized fragment is contacted with aBAV genome and, through homologous recombination or other conventionalgenetic engineering methods, the desired recombinant is obtained. Thegeneral methodology is described, for example, in Chartier et al. (1996)J. Virol 70:4805-4810. Recombination between the expression cassette anda BAV genome can occur within an appropriate helper cell line such as,for example, a procaryotic cell or an E1-transformed cell line, such asthat described by Graham et al. (1991) In Methods in Molecular Biology,Vol. 7, Humana Press, pp. 109-128. Heterologous sequences can also beintroduced into the BAV genome at sites other than the E1 and E3regions. It is within the skill of the art to isolate a restrictionfragment bearing a region of the BAV genome into which insertion ofheterologous sequences is desired, to clone heterologous sequences intosuch an isolated fragment of the BAV genome, and to reintroduce anisolated BAV fragment containing heterologous sequences into a BAVgenome to generate a BAV vector, either before or after transformationor transfection of an appropriate host cell.

[0051] Suitable host cells for construction of a BAV vector include anycell susceptible to transfection by BAV sequences (including a BAVgenome) that will support recombination between a BAV genome and aplasmid containing BAV sequences (optionally comprising heterologoussequences), or between two or more plasmids, each containing BAVsequences (one or both of which optionally comprises heterologoussequences). Recombination is preferably performed in procaryotic cells,such as E. coli, while transfection of a viral genome (optionallycontained in a plasmid) to generate virus particles is conducted ineukaryotic cells, preferably mammalian cells, more preferably bovinecells, still more preferably MDBK or PFBR cells, most preferably VIDO-R2cells. The growth of bacterial cell cultures, as well as culture andmaintenance of eukaryotic cells and mammalian cell lines are procedureswhich are well-known to those of skill in the art.

[0052] One or more heterologous sequences can be inserted into one ormore regions of a BAV genome to generate a recombinant BAV vector,limited only by the insertion capacity of the BAV genome and ability ofthe recombinant BAV vector to express the inserted heterologoussequences. Regions of the BAV genome suitable for insertion ofheterologous sequences include part or all of the E3 region, part or allof the E1 region, part or all of the E2 region, part or all of the E4region, part or all of the region between E4 and the right end of thegenome, part or all of the late regions (L1-L7) and/or part or all ofthe regions occupied by the 33 kD, 52 kD, 100 kD, DBP, pol, pTP andpenton genes, and genes IIIA, pV, pVI, pVII, pVIII and pX. In general,adenovirus genomes of approximately 105% of normal genome length remaincapable of being packaged into virus particles. The insertion capacitycan be increased by deletion of non-essential regions and/or deletion ofessential regions whose function is provided by a helper cell line or ahelper virus. Accordingly, the insertion capacity of a vector can dependupon the nature and extent of the viral function(s) provided in trans:in the sense that the greater the number of essential viral functionsprovided by the helper cell line or helper virus, the larger the portionof the vector genome that can be deleted; hence, the higher the capacityof the vector.

[0053] In one embodiment of the invention, insertion can be achieved byconstructing a plasmid containing the region of the BAV genome intowhich insertion is desired. The plasmid is then digested with one ormore restriction enzymes having a recognition sequence in the BAVportion of the plasmid, and a heterologous sequence is inserted at thesite of restriction digestion. The plasmid (or a linear fragment),containing a portion of the BAV genome with an inserted heterologoussequence, is co-transformed, along with a BAV genome or a linearizedfragment containing a BAV genome, into a bacterial cell (such as, forexample, E. coli), wherein the BAV genome can be a full-length genome orcan contain one or more deletions. Homologous recombination between theplasmids (and/or fragments) generates a plasmid (or a fragment)comprising a recombinant BAV genome containing inserted heterologoussequences.

[0054] In another embodiment of the invention, a recombinant expressioncassette can be obtained by cleaving a BAV genome with an appropriaterestriction enzyme to produce a DNA fragment representing the left endor the right end of the genome comprising E1 or E3 sequences,respectively, and inserting the left- or right-end fragment into acloning vehicle, such as a plasmid, and thereafter inserting at leastone heterologous DNA sequence into the E1 or E3 sequence, theheterologous sequence optionally in operative linkage with an exogenoustranscriptional regulatory sequence. The recombinant expression cassetteis contacted with a BAV genome within an appropriate cell and, throughhomologous recombination or other conventional genetic engineeringmethods, a recombinant BAV genome is obtained. Appropriate cells includeboth prokaryotic cells, such as, for example, E. coli, and eukaryoticcells. Examples of suitable eukaryotic cells include, but are notlimited to, MDBK cells, MDBK cells expressing adenovirus E1 function,primary fetal bovine retina cells, primary fetal bovine retina cellsexpressing adenovirus E1 function (such as VIDO-R2 cells) and cellsexpressing functions that are equivalent to those of thepreviously-recited cells.

[0055] Restriction fragments of the BAV genome other than thosecomprising the E1 and E3 regions are also useful in the practice of theinvention and can be inserted into a cloning vehicle such thatheterologous sequences may be inserted into sequences other that the E1and E3 regions. These DNA constructs can then undergo recombination invitro or in vivo, with a BAV genome, either before or aftertransformation or transfection of a suitable host cell as describedabove. For the purposes of the present invention, a BAV genome can beeither a full-length genome or a genome containing a deletion in aregion other than that deleted in the fragment with which it recombines,as long as the resulting recombinant BAV genome contains BAV sequencesrequired for replication and packaging. Methods for transfection, cellculture and recombination in procaryotic and eukaryotic cells such asthose described above are well-known to those of skill in the art.

[0056] Deletion of BAV sequences, to provide a site for insertion ofheterologous sequences or to provide additional capacity for insertionat a different site, can be accomplished by methods well-known to thoseof skill in the art. For example, for BAV sequences cloned in a plasmid,digestion with one or more restriction enzymes (with at least onerecognition sequence in the BAV insert) followed by ligation will, insome cases, result in deletion of sequences between the restrictionenzyme recognition sites. Alternatively, digestion at a singlerestriction enzyme recognition site within the BAV insert, followed byexonuclease treatment, followed by ligation will result in deletion ofBAV sequences adjacent to the restriction site. A plasmid containing oneor more portions of the BAV genome with one or more deletions,constructed as described above, can be co-transfected into a bacterialcell along with a BAV genome (full-length or deleted) or a plasmidcontaining either a full-length or a deleted BAV genome to generate, byhomologous recombination, a plasmid containing a recombinant BAV genomewith a deletion at one or more specific sites. BAV virions containingthe deletion can then be obtained by transfection of mammalian cells(including, but not limited to, MDBK, PFBR and VIDO-R2 cells and theirequivalents) with the plasmid containing the recombinant BAV genome.

[0057] In one embodiment of the invention, insertion sites are adjacentto and downstream (in the transcriptional sense) of BAV promoters.Locations of BAV promoters, and restriction enzyme recognition sequencesdownstream of BAV promoters, for use as insertion sites, can be easilydetermined by one of skill in the art from the BAV nucleotide sequenceprovided in co-owned International Patent Applications PCT/CA94/00678and PCT/CA98/00624. Alternatively, various in vitro techniques can beused for insertion of a restriction enzyme recognition sequence at aparticular site, or for insertion of heterologous sequences at a sitethat does not contain a restriction enzyme recognition sequence. Suchmethods include, but are not limited to, oligonucleotide-mediatedheteroduplex formation for insertion of one or more restriction enzymerecognition sequences (see, for example, Zoller et al. (1982) NucleicAcids Res. 10:6487-6500; Brennan et al. (1990) Roux 's Arch. Dev. Biol.199:89-96; and Kunkel et al. (1987) Meth. Enzymology 154:367-382) andPCR-mediated methods for insertion of longer sequences. See, forexample, Zheng et al. (1994) Virus Research 31:163-186.

[0058] It is also possible to obtain expression of a heterologoussequence inserted at a site that is not downstream from a BAV promoter,if the heterologous sequence additionally comprises transcriptionalregulatory sequences that are active in eukaryotic cells. Suchtranscriptional regulatory sequences can include cellular promoters suchas, for example, the bovine hsp70 promoter and viral promoters such as,for example, herpesvirus, adenovirus and papovavirus promoters and DNAcopies of retroviral long terminal repeat (LTR) sequences.

[0059] In another embodiment, homologous recombination in a procaryoticcell can be used to generate a cloned BAV genome comprising an E1deletion; and the cloned, E1-deleted BAV genome can be propagated as aplasmid. Infectious virus can be obtained by transfection of VIDO-R2cells, or their equivalents, with the cloned, E1-deleted BAV genomerescued from plasmid-containing cells.

[0060] The host cells of the invention, which provide essential viralfunctions, can be used for expression of proteins or peptides encoded byheterologous sequences included in recombinant BAV vectors. Methods forexpression and purification of recombinant proteins and peptides arewell-known to those of skill in the art; e.g., Ausubel et al., supra.

[0061] Additional methods for preparation of recombinant adenoviralgenomes, including BAV genomes, by recombination in a procaryotic cell,and transformation of mammalian cells (including bovine cells) with therecombinant genomes so generated, to generate recombinant adenovirusvectors, are described in co-owned International applicationsPCT/CA94/00678 and PCT/CA98/00624, the disclosures of which are herebyincorporated by reference in their entireties.

[0062] E. Therapeutic Genes and Polypeptides

[0063] BAV vectors that are propagated using the cells and cell lines ofthe invention can be used for the expression of therapeutic polypeptidesand nucleic acids in applications such as in vitro polypeptideproduction, vaccine production, nucleic acid immunization and genetherapy, for example.

[0064] In one embodiment, BAV vectors propagated in the host cells ofthe invention will contain heterologous sequences encoding protectivedeterminants of various mammalian pathogens, for use in subunit vaccinesand nucleic acid immunization. Representative mammalian pathogenantigens include, but are not limited to, bacterial pathogens, such asPasteurella sp and Hemophilus sp.; and viral pathogens, such asherpesviruses, influenzaviruses, parainfluenzaviruses, rotaviruses,coronaviruses, viral diarrhea viruses, picornaviruses, adenoviruses,retroviruses, lentiviruses, etc. BAV vectors can also include genesencoding cytokines, such as interferons, interleukins andcolony-stimulating factors (either instead of or in addition tosequences encoding protective determinants) and therapeutic polypeptidessuch as the cystic fibrosis transmembrane conductance regulator (CFTR)and coagulation factor IX, for example.

[0065] Various foreign genes or nucleotide sequences or coding sequences(prokaryotic or eukaryotic) can be inserted into a BAV vector that ispropagated in accordance with the present invention, particularly toprovide protection against a wide range of diseases. Protection can beprovided by way of subunit vaccines, nucleic acid immunization and/orgene therapy, using recombinant vectors propagated according to themethods of the invention.

[0066] A heterologous (i.e., foreign) nucleotide sequence can consist ofone or more gene(s) of interest, and preferably of therapeutic interest.In the context of the present invention, a gene of interest can encode astructural RNA, a ribosomal RNA, an antisense RNA, a ribozyme or it canencode an mRNA which will then be translated into a protein of interest.A gene of interest can be of genomic type, of complementary DNA (cDNA)type or of mixed type (i.e., a minigene, in which at least one intron isdeleted). It can code for a mature protein; a precursor of a matureprotein, in particular a precursor intended to be secreted andaccordingly comprising a signal peptide; a chimeric protein originatingfrom the fusion of sequences of diverse origins; or a mutant of anatural protein displaying improved or modified biological properties.Such a mutant can be obtained by deletion, substitution and/or additionof one or more nucleotide(s) of the gene coding for the natural protein,or any other type of change in the sequence encoding the naturalprotein, such as, for example, transposition or inversion.

[0067] A gene of interest can be placed under the control of regulatorysequences suitable for its expression in a host cell. Suitableregulatory sequences are understood to mean the set of elements neededfor transcription of a gene into RNA (structural, ribosomal, ribozyme,antisense RNA or mRNA), for processing of RNA, and for the translationof an mRNA into protein. Among the elements needed for transcription,the promoter assumes special importance. It can be a constitutivepromoter or a regulatable promoter, and can be isolated from any gene ofeukaryotic, prokaryotic or viral origin, and even adenoviral origin.Alternatively, it can be the natural promoter of the gene of interest.Generally speaking, a promoter used in the present invention can bechosen to contain cell-specific regulatory sequences, or modified tocontain such sequences. For example, a gene of interest for use in thepresent invention is placed under the control of an immunoglobulin genepromoter when it is desired to target its expression to lymphocytic hostcells. There may also be mentioned the HSV-1 TK (herpesvirus type 1thymidine kinase) gene promoter, the adenoviral MLP (major latepromoter), in particular of human adenovirus type 2, the RSV (RousSarcoma Virus) LTR (long terminal repeat), the CMV (Cytomegalovirus)early promoter, and the PGK (phosphoglycerate kinase) gene promoter, forexample, permitting expression in a large number of cell types.

[0068] In addition to promoters, enhancer sequences are also importantin regulating the degree of expression of a gene or coding sequence towhich they are operatively linked. A heterologous gene or codingsequence can be regulated by an endogenous adenoviral enhancer presentin the vector, or can be regulated by a non-vector enhancer. Anon-vector enhancer can be an enhancer normally associated with theheterologous gene in its natural state, or one that is not normallyassociated with the gene or coding sequence, but is placed in operativelinkage with the gene or coding sequence by in vitro techniques.

[0069] Alternatively, targeting of a recombinant BAV vector to aparticular cell type can be achieved by constructing recombinant hexonand/or fiber genes. The protein products of these genes are involved inhost cell recognition; therefore, the genes can bc modified to containpeptide sequences that will allow the virus to recognize alternativehost cells.

[0070] Among genes of interest which arc useful in the context of thepresent invention, there may be mentioned:

[0071] genes coding for cytokines such as interferons and interleukins;

[0072] genes encoding lymphokines;

[0073] genes coding for membrane receptors such as the receptorsrecognized by pathogenic organisms (viruses, bacteria or parasites),preferably by the HIV virus (human immunodeficiency virus);

[0074] genes coding for coagulation factors such as factor VIII andfactor IX;

[0075] genes coding for dystrophins;

[0076] genes coding for insulin;

[0077] genes coding for proteins participating directly or indirectly incellular ion channels, such as the CFTR (cystic fibrosis transmembraneconductance regulator) protein;

[0078] genes coding for antisense RNAs, or proteins capable ofinhibiting the activity of a protein produced by a pathogenic gene whichis present in the genome of a pathogenic organism, or proteins (or genesencoding them) capable of inhibiting the activity of a cellular genewhose expression is deregulated, for example an oncogene;

[0079] genes coding for a protein inhibiting an enzyme activity, such asα₁-antitrypsin or a viral protease inhibitor, for example;

[0080] genes coding for variants of pathogenic proteins which have beenmutated so as to impair their biological function, such as, for example,trans-dominant variants of the tat protein of the HIV virus which arecapable of competing with the natural protein for binding to the targetsequence, thereby preventing the activation of HIV;

[0081] genes coding for antigenic epitopes in order to increase the hostcell's immunity;

[0082] genes coding for major histocompatibility complex classes I andII proteins, as well as the genes coding for the proteins which areinducers of these genes;

[0083] genes coding for antibodies;

[0084] genes coding for immunotoxins;

[0085] genes encoding toxins;

[0086] genes encoding growth factors or growth hormones;

[0087] genes encoding cell receptors and their ligands;

[0088] genes encoding tumor suppressors;

[0089] genes coding for cellular enzymes or those produced by pathogenicorganisms; and

[0090] suicide genes. The HSV-1 TK suicide gene may be mentioned as anexample. This viral TK enzyme displays markedly greater affinitycompared to the cellular TK enzyme for certain nucleoside analogues(such as acyclovir or gancyclovir). It converts them tomonophosphorylated molecules, which can themselves be converted bycellular enzymes to nucleotide precursors, which are toxic. Thesenucleotide analogues can be incorporated into replicating DNA molecules,hence incorporation occurs chiefly in the DNA of dividing cells. Thisincorporation can result in specific destruction of dividing cells suchas cancer cells.

[0091] Although any gene or coding sequence of therapeutic relevance canbe used in the practice of the invention, certain genes, or fragmentsthereof, are particularly suitable. For example, genes encodingimmunogenic polypeptides, toxins, immunotoxins and cytokines are usefulin the practice of the invention. Cytokine genes of use in the inventioninclude, but are not limited to, those encoding α, βor γ interferon(IFN), interleukins (IL) such as IL-2, IL-6, IL-10 or IL-12, tumornecrosis factor (TNF), colony stimulating factors such as GM-CSF, C-CSF,M-CSF, and other cytokines as are known to those of skill in the art.Additional genes include those encoding cell or nuclear receptors andtheir ligands (e.g.,fas ligand), coagulation factors (for example,FVIII, FIX), growth hormones, growth factors such as fibroblast growthfactors (FGF), vascular endothelial growth factors (VEGF), nerve growthfactors (NGF), epidermal growth factors (EGF), platelet-derived growthfactors (PDGF) and other growth factors as are known to those of skillin the art. Genes suitable for use in the practice of the invention canencode enzymes (such as, for example, urease, renin, thrombin,metalloproteases, nitric oxide synthase, superoxide dismutase, catalaseand others known to those of skill in the art), enzyme inhibitors (suchas, for example, α1-antitrypsin, antithrombin III, cellular or viralprotease inhibitors, plasminogen activator inhibitor-1, tissue inhibitorof metalloproteases, etc.), the cystic fibrosis transmembraneconductance regulator (CFTR) protein, insulin, dystrophin, or a MajorHistocompatibility Complex (MHC) antigen of class I or II. Also usefulare genes encoding polypeptides that can modulate/regulate expression ofcorresponding genes, polypeptides capable of inhibiting a bacterial,parasitic or viral infection or its development (for example, antigenicpolypeptides, antigenic epitopes, and transdominant protein variantsinhibiting the action of a native protein by competition), apoptosisinducers or inhibitors (for example, Bax, Bcl2, BclX and others known tothose of skill in the art), cytostatic agents (e.g., p21, p16, Rb,etc.), apolipoproteins (e.g., ApoAI, ApoAIV, ApoE, etc.), angiogenesisinhibitors (e.g, angiostatin, endostatin, etc.), oxygen radicalscavengers, polypeptides having an anti-tumor effect, antibodies,toxins, immunotoxins, markers (e.g., β-galactosidase, luciferase, etc.)or any other genes of interest that are recognized in the art as beinguseful for treatment or prevention of a clinical condition.

[0092] For example, with respect to treating hereditary dysfuncitons,one may use a functional copy of a defective gene, for example a geneencoding factor VIII or IX in the context of haemophilia A or B,dystrophin (or minidystrophin) in the context of myopathies, insulin inthe context of diabetes, or CFTR (Cystic Fibrosis TransmembraneConductance Regulator) in the context of cystic fibrosis. Suitable genesof interest to delay or inhibit tumor or cancer progression, include butare not limited to those encoding an antisense RNA, a ribozyme, acytotoxic product such as thymidine kinase of herpes simplex virus type1 (HSV-1TK), ricin, a bacterial toxin, the products of the yeast genesFCY1 and/or FUR1 having CDase (cytosine deaminase) and UPRTase (uracilphosphoribosyl transferase) activities respectively, an antibody, apolypeptide inhibiting cellular division or signal transduction, a tumorsuppressor gene (such as, for example, p53, Rb, p73), a polypeptidewhich activates the host immune system, a tumor-associated antigen (e g,MUC-1, BRCA-1, an HPV early or late antigen such as E6, E7, L1, L2,etc), optionally in combination with a cytokine gene. Finally, in thecontext of anti-HIV therapy, one may use a gene encoding animmunoprotective polypeptide, an antigenic epitope, an antibody (2F5;Buchacher et al., 1992, Vaccines 92:191-195), the extracellular domainof CD4 (sCD4; Traunecker et al., 1988, Nature 331:84-86), animmunoadhesin (i e., a CD4-IgG hybrid, CD4-2F5 fusion; Capon et al.,1989, Nature 337:525-531; Byrn et al., 1990, Nature 344:667-670), animmunotoxin (i.e., resulting from fusion between angiogenin and 2F5 orCD4-2F5; Kurachi et al., 1985, Biochemistry 24:5494-5499), atrans-dominant variant (EP 0614980, W095/16780), a cytotoxic product(see above) or IFNα or β.

[0093] In addition, a gene of interest may also encode all or part of aselective marker, allowing the selection of transfected and transducedcells. Such genes include but are not limited to the neo gene (encodingneomycin phosphotransferase) conferring resistance to G418, dhfr(Dihydrofolate Reductase), CAT (Chloramphenicol Acetyl Transferase), pac(Puromycin Acetyl-Transferase) and gpt (Xanthine Guanine PhosphoribosylTransferase). Genes encoding selective markers are known in the art.

[0094] The above-mentioned genes and coding regions, as well as othersknown to those of skill in the art, are suitable for use in any aspectof the invention, including protein production, vaccination, nucleicacid immunization and/or gene therapy, among others.

[0095] This above list is not restrictive, and any other gene ofinterest can be used in the context of the present invention. In somecases the gene for a particular antigen can contain a large number ofintrons or can be from an RNA virus, in these cases a complementary DNAcopy (cDNA) of the gene transcript or of the viral genome can be used.It is also possible that only fragments of nucleotide sequences of genescan be used (where these are sufficient to generate a protective immuneresponse or a specific biological effect) rather than the completesequence as found in the wild-type organism. Where available, syntheticgenes or fragments thereof can also be used. However, the presentinvention can be used with a wide variety of genes, fragments and thelike, and is not limited to those set out above.

[0096] Recombinant BAV vectors propagated in the host cells of theinvention can be used to express antigens for provision of, for example,subunit vaccines. Antigens used in the present invention can be eithernative or recombinant antigenic polypeptides or fragments. They can bepartial sequences, full-length sequences, or fusions (c.g., havingappropriate leader sequences for the recombinant host, or with anadditional antigen sequence for another pathogen). The preferredantigenic polypeptide to be expressed by the virus systems of thepresent invention contains full-length (or near full-length) sequencesencoding antigens. Alternatively, shorter sequences that are antigenic(i.e., encode one or more epitopes) can be used. The shorter sequencecan encode a “neutralizing epitope,” which is defined as an epitopecapable of eliciting antibodies that neutralize virus infectivity in anin vitro assay. Preferably the peptide should encode a “protectiveepitope” that is capable of raising in the host a “protective immuneresponse;” i.e., a humoral (i.e. antibody-mediated), cell-mediated,and/or mucosal immune response that protects an immunized host frominfection.

[0097] F. Therapeutic Applications

[0098] With the recombinant viruses produced using the host cells of thepresent invention, it is possible to provide protection against a widevariety of diseases affecting mammals. Any of the recombinant antigenicdeterminants or recombinant live virus vectors propagated according tothe methods of the invention can be formulated and used in substantiallythe same manner as described for antigenic determinant vaccines or livevaccine vectors.

[0099] Antigens expressed by vectors propagated according to the methodsof the present invention, particularly when comprised of shortoligopeptides, can be conjugated to a vaccine carrier. Vaccine carriersare well known in the art: for example, bovine serum albumin (BSA),human serum albumin (HSA) and keyhole limpet hemocyanin (KLH). Apreferred carrier protein, rotavirus VP6, is disclosed in EPO Pub. No.0259149, the disclosure of which is incorporated by reference herein.

[0100] Genes for desired antigens or coding sequences thereof which canbe inserted include those of organisms which cause disease in mammals,particularly bacterial and viral pathogens. Genes encoding antigens ofhuman pathogens are also useful in the practice of the invention.Representative mammalian pathogen antigens include, but are not limitedto, bacterial pathogens, such as Pasteurella sp and Hemophilus sp.; andviral pathogens, such as herpesviruses, influenzaviruses,parainfluenzaviruses, rotaviruses, coronaviruses, viral diarrheaviruses, picomaviruses, adenoviruses, retroviruses, lentiviruses, etc.BAV vectors can also include genes encoding cytokines, such asinterferons, interleukins and colony-stimulating factors (either insteadof or in addition to sequences encoding protective determinants) andtherapeutic polypeptides such as the cystic fibrosis transmembraneconductance regulator (CFTR) and coagulation factor IX, for example.

[0101] The present invention also includes pharmaceutical compositionscomprising a therapeutically effective amount of a recombinant vector,recombinant virus or recombinant protein, prepared according to themethods of the invention, in combination with a pharmaceuticallyacceptable vehicle and/or an adjuvant. Such a pharmaceutical compositioncan be prepared and dosages determined according to techniques that arewell-known in the art. The pharmaceutical compositions of the inventioncan be administered by any known administration route including, but notlimited to, systemically (for example, intravenously, intratracheally,intraperitoneally, intranasally, parenterally, enterically,intramuscularly, subcutaneously, intratumorally or intracranially) or byaerosolization or intrapulmonary instillation. Administration can takeplace in a single dose or in doses repeated one or more times aftercertain time intervals. The appropriate administration route and dosagewill vary in accordance with the situation (for example, the individualbeing treated, the weight of the individual, the disorder to be treatedor the gene or polypeptide of interest), but can be determined by one ofskill in the art.

[0102] The vaccines of the invention carrying foreign genes or fragmentscan be orally administered in a suitable oral carrier, such as in anentcric-coated dosage form. Oral formulations include suchnormally-employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearatc, sodium saccharincellulose, magnesium carbonate, and the like. Oral vaccine compositionsmay be taken in the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations, or powders, containing fromabout 10% to about 95% of the active ingredient, preferably about 25% toabout 70%. An oral vaccine may be preferable to raise mucosal immunity(which plays an important role in protection against pathogens infectingthe gastrointestinal tract) in combination with systemic immunity.

[0103] In addition, the vaccine can be formulated into a suppository.For suppositories, the vaccine composition will include traditionalbinders and carriers, such as polyalkaline glycols or triglycerides.Such suppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%.

[0104] Protocols for administering to mammals the vaccine composition(s)of the present invention are within the skill of the art in view of thepresent disclosure. Those skilled in the art will select a concentrationof the vaccine composition in a dose effective to elicit antibody,cell-mediated and/or mucosal immune responses to the antigenic fragment.Within wide limits, the dosage is not believed to be critical.Typically, the vaccine composition is administered in a manner whichwill deliver between about 1 to about 1,000 micrograms of the subunitantigen in a convenient volume of vehicle, e.g., about 1-10 ml.Preferably, the dosage in a single immunization will deliver from about1 to about 500 micrograms of subunit antigen, more preferably about 5-10to about 100-200 micrograms (e.g., 5-200 micrograms).

[0105] The timing of administration may also be important. For example,a primary inoculation preferably may be followed by subsequent boosterinoculations, for example, several weeks to several months after theinitial immunization, if needed. To insure sustained high levels ofprotection against disease, it may be helpful to readminister boosterimmunizations at regular intervals, for example once every severalyears. Alternatively, an initial dose may be administered orallyfollowed by later inoculations, or vice versa. Preferred vaccinationprotocols can be established through routine vaccination protocolexperiments.

[0106] A problem that has beset the use of adenovirus vectors forimmunization and gene therapy in humans is the rapid development of animmunological response (or indeed in some cases existing immunity) tohuman adenoviruses (HAds). Recombinant BAV vectors are likely to be lessimmunogenic in humans and, for this and other reasons, will be usefuleither as a substitute for HAd vectors or in combination with HAdvectors. For example, an initial immunization with a HAd vector can befollowed by booster immunizations using BAV vectors; alternatively,initial immunization with a recombinant BAV vector can be followed bybooster immunizations with HAd and/or BAV vectors.

[0107] The dosage for all routes of administration of in vivorecombinant virus vaccine depends on various factors including, the sizeof patient, nature of infection against which protection is needed,carrier and the like and can readily be determined by those of skill inthe art. By way of non-limiting example, a dosage of betweenapproximately 10³ pfu and 10¹³ pfu, preferably 10³ to 10¹⁰ pfu, morepreferably, 10³ to 10⁸ pfu can be used. As with in vitro subunitvaccines, additional dosages can be given as determined by the clinicalfactors involved.

[0108] The invention also encompasses a method of treatment, accordingto which a therapeutically effective amount of a BAV vector, recombinantBAV, or host cell of the invention is administered to a mammaliansubject requiring treatment.

[0109] G. Gene Therapy

[0110] Recombinant adenovirus vectors and their genomes, produced usingthe host cells of the invention, can be used in methods for providinggene therapy to a mammal, to control a gene deficiency. In oneembodiment, these methods comprise administering to said mammal a liverecombinant BAV containing a heterologous nucleotide sequence encoding anon-defective form of a deficient gene, under conditions wherein therecombinant virus vector genome is incorporated into the mammaliangenome or is maintained independently and extrachromosomally, to provideexpression of the non-defective gene in a particular target organ ortissue. These and related techniques can also be used to replace adefective gene or portion thereof. Non-limiting examples of foreigngenes, heterologous nucleotide sequences, or portions thereof that canbe incorporated for use in gene therapy have been discussed above insection E, entitled “Therapeutic genes and polypeptides.”

[0111] In particular, use of adenovirus vectors propagated in the hostcells of the invention in regard to gene therapy in humans is intendedfor the prevention or treatment of diseases including, but not limitedto, genetic diseases (for example, hemophilia, thalassemias, myopathies,muscular dystrophy, diabetes, emphysema, Gaucher's disease, cysticfibrosis, Duchenne muscular dystrophy, Duchenne's or Becker's myopathy,etc.), cancers, viral diseases (for example, AIDS, herpesvirusinfection, Cytomegalovirus infection and papillomavirus infection),immune deficiency diseases, cardiovascular diseases, and the like. Forthe purposes of the present invention, the vectors, cells and viralparticles prepared by the methods of the invention can be introducedinto a subject either ex vivo, (i.e., in a cell or cells removed fromthe patient) or directly in vivo into the body to be treated, into anytype of cell. Preferably, the host cell is a human cell and, morepreferably, is a lung cell, an airway epithelial cell, a fibroblast, amuscle cell (including smooth muscle, striated muscle and cardiacmuscle), a liver cell, a lymphocytic cell, a cell of the hematopoieticlineage, an endothelial cell or a malignantly transformed descendant ofthese or any other cell.

[0112] Described below are examples of the present invention. Theseexamples are provided for illustrative purposes only and are notintended to limit the scope of the present invention in any way. Inlight of the present disclosure, numerous embodiments within the scopeof the claims will be apparent to those of ordinary skill in the art.The contents of the references cited in the specification areincorporated by reference herein.

EXAMPLES

[0113] General Methods

[0114] BAV-3 was cultured in Madin-Darby bovine kidney (MDBK) cells orVIDO R2 cells grown in Eagle's minimal essential medium supplementedwith 5% fetal bovine serum. Viral DNA was extracted from virus-infectedcell monolayers by the method of Hirt (1967) J. Mol. Biol. 26:365-369.Recombinant plasmids were constructed by standard proceudures usingrestriction endonucleases and other DNA modifying enzymes according tothe manufacturers' instructions.

Example 1 Construction and Properties of the VIDO-R2 Cell Line

[0115] Primary cultures of fetal bovine retina cells (FBRC) weretransfected with 10 μg of plasmid pTG4671 (Transgene) by calciumphosphate co-precipitation. This plasmid contains the entire E1A and E1Bsequences (nucleotides 505-4034) of human adenovirus-5 (GenBankAccession No. M73260), with E1A transcription under the control of theconstitutive mouse phosphoglycerate kinase promoter and E1 Btranscription under the control of its natural promoter and a β-globinpolyadenylation signal. Chroboczek et al. (1992) Virology 186:280-285;Adra et al. (1987) Gene 60:65-74. A gene encoding the selectable markerpuromycin acetyl transferase (pac), under the control of theconstitutive SV40 early promoter and a SV40 polyadenylation signal, isalso present in plasmid TG4671. See FIG. 1.

[0116] Transformed cells were cultured without selection for puromycinresistance. Four weeks after transfection, foci of transformed cellswere observed. The transformed cells were smaller and rounder thanuntransformed cells. See FIG. 2. Transformed cells expressed vimentin,but not cytokeration, indicating that they are of mesenchymal origin.Cell foci were subjected to single cell cloning. One of the clonesobtained was named VIDO R2.

[0117] Expression of E1 mRNA was examined by reverse transcriptionpolymerase chain reaction (RT-PCR) analysis, using pairs of primersspecific for the E1A- and E1B regions of HAV-5. RT-PCR using R2 cell RNAgenerated products that matched the size of PCR products generated froman E1 DNA template using the same primers. When reverse transcriptasewas omitted from the RT-PCR reaction using R2 cell RNA, no product wasobserved, indicating that the amplification products were derived fromE1 mRNAs and not residual DNA.

[0118] Expression of E1A and E1B proteins was analyzed by immunologicalanalysis of protein blots (Western blotting), using mouse monoclonalantibody M73 to detect E1A proteins, and antibody 3D11 (Calbiochem, LaJolla, Calif.) to detect the 19kDa HAV E1B protein. Both E1A (FIG. 3)and E1B (FIG. 4) polypeptides were produced by the VIDO-R2 cell line,and were not detected in the parental FBRC line.

[0119] Doubling time in cell culture was also determined for the R2 cellline. Visual inspection of cultures showed that VIDO-R2 cells formedmonolayers within 2-3 days after plating a 1:3 dilution of confluentcells, while the parent PFBR cells required 10-15 days to formmonolayers under the same conditions. PCR experiments, using VIDO-R2cell genomic DNA as template, indicated that the E1 sequences present inVIDO-R2 were integrated into the cellular genome.

Example 2 Complementing properties of VIDO R2 cells

[0120] To investigate the complementing properties of the VIDO R2, thecells were infected with an E1A deletion mutant of HAV-5 (Ad5d1E1AlacZ).Zheng et al. (1994) Virus Res. 31:163-186. This cell line supported thegrowth of the deletion mutant to 10⁷ pfu/ml. To determine whether theVIDO R2 cell line could support plaque formation, cells cultured in35-mm-diameter dishes were infected with BAV-3 or HAV-5 and incubated ina CO₂ incubator. Clear plaque formation was evident on day 5 and 7postinfection with HAV-5 and BAV-3, respectively. A substantially morerapid onset of viral cytopathic effect was observed in E1-expressingcell lines as opposed to MDBK and FBRC lines. In addition, the R2 cellline supported formation of clear plaques by recombinant BAV-3.

Example 3 Transfection Ability of VIDO R2 Cells

[0121] To test the ability of the cells to take up large DNA, MDBK andVIDO R2 cells, in 35 mm-diameter dishes, were transfected with 1-3 ug ofPacI-restricted plasmid pFBAV304 using Lipofectin (GIBCO/BRL). Thisplasmid contains the entire BAV-3 genome with the E3 region replaced bya green fluorescent protein (GFP) gene under the control of acytomegalovirus immediate early promoter. See Example 7. When observedby fluorescence microscopy 24 hours following transfection, more than 3%of VIDO R2 cells showed fluorescence, as opposed to less than 0.1% ofthe cells in MDBK cultures. Further incubation of the transfected VIDOR2 cells for 10-14 days resulted in production of a recombinant virus(BAV 304) expressing GFP. These observations suggest that VIDO R2 cellsare well-suited for generation of recombinant BAV-3, perhaps in partowing to higher transfection efficiency and/or the presence of HAV-5 E1Aand E1B sequences.

Example 4 Construction of BAV3.500, a Replication-Defective, RecombinantBAV with Deletions in E1 and E3

[0122] A BAV3 with a genome having deletions in the E1A and E3 regionswas constructed as follows.

[0123] The plasmid pTG5435 (FIG. 5), comprising a full-length BAV-3genome in a ppolyIIsn14 plasmid backbone (Lathe et al. (1987) Gene57:193-201) was digested with HindIII, and a 4.9 kb fragment harboringthe terminal BAV-3 sequences was isolated and religated, creatingplasmid pLt-Rt.Hind (pBAV-101). The plasmid pLt-Rt.Hind was digestedwith AccI and SpeI, generating 2 fragments, which were treated with T4DNA polymerase to generate blunt ends. The larger fragment (4.4 kbp) wasisolated and ligated to a XbaI linker to create plasmid pLR.Hind-XbaI(pBAV-102), containing a deletion in E1 with the deleted sequencesreplaced by an XbaI site. This plasmid was digested with HindIII,dephosphorylated, and the linear dephosphorylated fragment wasgel-purified. The gel-purified fragment was recombined with genomic DNAof recombinant BAV.E3d (a BAV genome containing a 1.245 kb deletion inthe E3 region) by co-transformation of E. coli, to create plasmidpBAV3.500 (pFBAV500). See FIG. 6

[0124] PacI-digested pFBAV500 DNA was transfected into VIDO-R2 cells(5-10 μg per 60 mm diameter dish of cell monolayer) using Lipofectin(GIBCO/BRL). After incubation at 37° C., cells showing cytopathiceffects were collected and subjected to two cycles of freeze-thawing,and recombinant virus (BAV3.500) was plaque-purified on VIDO R2 cells.

Example 5 Construction of BAV3.501, a Replication-Defective RecombinantBAV with an Insertion of the Bovine Herpesvirus Type 1 Glycoprotein DGene in the E1A Region

[0125] A BAV3 genome containing deletions in the E1A and E3 regions,with an insertion of BHV-1 gD in place of the deleted E1A sequences wasconstructed as follows. See FIG. 7.

[0126] Plasmid pBAV-102 (See Example 4) was digested with Xba I, treatedwith T4 DNA polymerase, dephosphorylated and gel-purified. A blunt-ended1.8 kb fragment, containing the bovine herpesvirus type 1 (BHV-1)glycoprotein D (gD) gene, including a 137-nucleotide chimeric intron andflanked upstream by the SV40 early promoter and downstream by the SV40late polyadenylation site, was ligated to the gel-purified XbaI fragmentto create a plasmid. pLR.Hb.gD (pBAV-102gD), containing a deletion inE1A with the deleted sequences replaced by the BHV-1 gD gene. Thisplasmid was recombined with genomic DNA of recombinant BAV.E3d (a BAVgenome containing a 1.245 kb deletion in the E3 region) byco-transfection of E. coli BJ5183, to create plasmid pBAV3.501(pFBAV501).

[0127] PacI-digested pFBAV501 DNA was transfected into VIDO-R2 cells(5-10 μg per 60 mm diameter dish of cell monolayer) using Lipofectin(GIBCO/BRL) After incubation at 37° C., cells showing cytopathic effectswere collected and subjected to two cycles of freeze-thawing, andrecombinant virus (BAV3.501) was plaque-purified on VIDO R2 cells.

[0128] The presence of the gD insert was confirmed by Cla I digestion,which indicated the loss of a 2.5 kb fragment characteristic of BAV3.500and its replacement by a fragment of 4.4 kb. Southern blot analysis witha gD probe confirmed that gD sequences were present in the 4.4 kbfragment.

Example 6 Construction of BAV3.502, a Replication-Defective RecombinantBAV with an Insertion of the Bovine Coronavirus HE Gene in the E3 Region

[0129] A recombinant BAV genome, containing an insertion of the bovinecoronavirus (BCV) hemagglutinin-esterase (HE) gene in the E3 region, inthe same transcriptional orientation as E3, was constructed as follows.See FIG. 8.

[0130] The BCV HE gene insert contained a 137-nucleotide chimeric intronand was flanked by the SV40 early promoter and a SV40 polyadenylationsite. This recombinant genome was introduced into E. coli BJ5183, alongwith HindIII-digested pBAV-102. In vivo recombination between these twoDNA molecules generated pFBAV502.

[0131] PacI-digested pFBAV502 DNA was transfected into VIDO-R2 cells(5-10 μg per 60 mm diameter dish of cell monolayer) using Lipofectin(GIBCO/BRL). After incubation at 37° C., cells showing cytopathiceffects were collected and subjected to two cycles of freeze-thawing,and recombinant virus (BAV3.502) was plaque-purified on VIDO R2 cells.

[0132] The presence of the HE insert was confirmed by Cla I digestion,which indicated the loss of a 12.2 kb fragment characteristic ofBAV3.500 and its replacement by a fragment of 14.1 kb. Southern blotanalysis with a HE probe confirmed that HE sequences were present in the14.1 kb fragment.

Example 7 Construction of BAV3.304, a Recombinant BAV with an Insertionof the Green Fluorescent Protein Gene in the E3 Region

[0133] A green fluorescent protein (GFP) gene, under the control of thecytomegalovirus immediate early promoter and the bovine growth hormonepolyadenylation signal, was obtained from the plasmid pQBI 25 (QuantumBiotechnologies) by Bgl II and Dra III digestion followed by treatmentwith T4 DNA polymerase to generate blunt ends. This fragment was theninserted into the Srf I site of pBAV-301, with the GFP gene in the sametranscriptional orientation as E3, to generate pBAV-301.gfp.

[0134] pBAV-301 was constructed by ligating a 7,635 base-pair Kpn I-SspI fragment of pFBAV302 to Kpn I/Not I digested PpolyIIsn14. pFBAV302 isa BAV genome with with an E3 deletion in which the deleted E3 sequencesare replaced by a Srf I site.

[0135] A Kpn I/Sma I fragment of pBAV301.gfp, encompassing the modifiedE3 region, was introduced into E. coli BJ 5183, along with SrfI-digested pFBAV.302. In vivo recombination between these two DNAmolecules generated pFBAV.304, a BAV genome containing a GFP gene in adeleted E3 region. See FIG. 9.

[0136] PacI-digested pFBAV.304 DNA was transfected into VIDO-R2 cells(5-10 μg per 60 mm diameter dish of cell monolayer) using Lipofectin(GIBCO/BRL). After incubation at 37° C., cells showing cytopathiceffects were collected and subjected to two cycles of freeze-thawing,and recombinant virus (BAV3.304) was plaque-purified on VIDO R2 cells.

[0137] pFBAV.304 viral DNA was analyzed by Bam HI digestion followed byagarose gel electrophoresis. Bam HI digestion of pFBAV.304 produced a2.3 kb fragment that was not present in the parental BAV.E3d genome.Southern blot analysis with a GFP probe confirmed that GFP sequenceswere present in the 2.3 kb Bam HI fragment.

Example 8 Abortive Infection of Noncomplementing Cell Lines with E1Mutant Viruses

[0138] FBRC and R2 cells were infected with wild-type or recombinant BAVat a MOI of less than one, cultured for one week, subjected to twofreeze-thaw cycles, and titrated on VIDO R2 cells. Wild-type BAV-3, anE3 deletion (BAV-3.E3d) and BAV3.304 grew to high titers (up to 10⁹pfu/ml) in all cell lines tested, whereas replication-defectiverecombinant viruses containing deletions in E1 and E3 (BAV3.500,BAV3.501 and BAV3.502) grew only in VIDO R2 cells, generating titers ofapproximately 10⁷ pfu/ml. See FIG. 10.

Example 9 Kinetics of gD Expression from Recombinant Viruses

[0139] Kinetics of gD expression by BAV3.501 (Example 5) weredetermined, by immunoprccipitation, at three time points after infectionof VIDO R2 or MDBK cells (FIG. 11). For immunoprecipitation analysis,confluent monolayers of VIDO R2 cells in six-well dishes were infectedwith virus at a MOI of greater than 5. Cells were preincubated for 2 hin minimal essential medium lacking methionine and cysteine prior tolabeling for 4 h with 50 μCi of [³⁵S]methionine (Tran³⁵S-label,phosphate-buffered saline, 1,000 Ci/mmol, ICN Radiochemicals, Inc.Irvine, Calif.). The cells were washed with phosphate-buffered saline,harvested by scraping, then lysed with ice-cold modifiedradioimmunoprecipitation assay buffer. Radiolabeled proteins wereimmunoprecipitated with a pool of anti-BHV-1 gD monoclonal antibodies(Hughes et al. (1988) Arch. Virol. 103:47-60) and analyzed bySDS-polyacrylamide gel electrophoresis. After running, the gels weredried and labeled protein bands were visualized by autoradiography.

[0140] Electrophoretic analysis of metabolically labeledimmunoprecipitates from lysates of BAV3.501-infected VIDO R2 cellsrevealed immunoreactive proteins with molecular weights of approximately63 kDa and 71 kDa (FIG. 11A, lanes 5 and 6), corresponding tounglycosylated and glycosylated forms of gD, respectively. Thesemolecular weights correspond to those of authentic gD immunoprecipitatedfrom BHV-1-infected cell extracts (FIG. 11A, lane 3). No proteins ofcorresponding molecular weight were detected in mock-infected cells(FIG. 11A, lane 1) or BAV-3-infected cells (FIG. 11A, lane 2).

[0141] In BAV3.501-infected cells, expression of gD was first detected24 hours after infection (FIG. 11 A, lane 5) and it continued to beproduced up to 36 hours post-infection (FIG. 11A, lane 6), which was thefinal time point used in the study. Kinetics of gD expression fromBAV3.501 were similar in MDBK cells (FIG. 11B, lanes 5 and 6).

Example 10 Kinetics of HE Expression from Recombinant Viruses

[0142] Kinetics of HE expression by BAV3.502 (Example 6) were determinedin VIDO R2 cells (FIG. 12). Immunoprecipitation analysis was conductedas described in Example 10, except that rabbit polyclonal anti-BCVantibodies were used for immunoprecipitation. Deregt et al. (1987)Virology 161:410-420; and Deregt et al. (1989) J. Gen. Virol.70:993-1998.

[0143] Anti-BCV polyclonal rabbit serum immunoprecipitated a 65 kDapolypeptide from R2 cells infected with BAV3.502 (lanes 5 and 6). Thispolypeptide comigrated with authentic HE protein produced fromBCV-infected cells (lane 3), and no corresponding protein wasimmunoprecipitated from mock-infected cells (lane 1) or from wild-typeBAV-3-infected cells (lane 2). Kinetics of HE expression (lanes 5 and 6)were similar to those observed for gD in BAV3.501-infected cells.

Example 11 Glycosylation of Recombinant gD and HE Proteins

[0144] Glycosylation of recombinant gD and HE proteins was examined byimmunoprecipitation following labeling of infected cells with [³H]glucosamine. Results of these studies confirmed that the proteinsproduced by recombinant bovine adenoviruses are glycosylated and areindistinguishable in migration rate (on gels) from the authenticproteins synthesized in virus-infected cells.

Example 12 Expression of GFP in BAV3.304-Infected Cells

[0145] Lysates of cells infected with BAV3.304 were examined by proteinblotting using GFP-specific polyclonal antibodies (Clontech, Palo Alto,Calif.). See FIG. 13. Cell extracts (5 μg per lane) were separated on a10% polyacrylamide-SDS gel and the gel weas blotted onto anitrocellulose membrane. Nonspecific binding sites on the membrane wereblocked with 1% bovine serum albumin and the blots were incubated withanti-GFP polyclonal antibodies. After antibody binding, the blots werewashed and exposed to anti-mouse or anti-rat IgG conjugated tohorseradish peroxidase (HRP) or alkaline phosphatase (AP), and developedusing HRP or AP development kits (Bio-Rad, Hercules, Calif.).

[0146] Anti-GFP serum identified a protein of 28 kDa inBAV3.304-infected cells (lanes 1-3) that was not present inmock-infected (lane 4) or wild-type BAV-infected cells (lane 5).Recombinant GFP was detected between 12 and 36 hours after infection(lanes 1-3).

[0147] Deposit of Biological Materials

[0148] The following materials were deposited and are maintained withthe American Type Culture Collection, Gaithersburg, Md.

[0149] Recombinant cell lines

[0150] Primary fetal bovine retinal cells transformed with HAd-5 E1sequences: Material Accession No. Deposit Date VIDO R2 ATCC PTA-156 June1, 1999

[0151] While the foregoing invention has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatvarious changes and modifications may be practiced without departingfrom the spirit of the invention. Therefore the foregoing descriptionsand examples should not be construed as limiting the scope of theinvention.

What is claimed is:
 1. A bovine cell that expresses a function essentialfor replication of an adenovirus, wherein the cell is permissive for thereplication of a recombinant adenovirus vector having a mutation in aregion of its genome corresponding to the essential function provided bythe host cell.
 2. The cell of claim 1 wherein the essential adenoviralfunction is E1 function.
 3. The cell of claim 2 wherein the genome ofthe recombinant adenovirus vector is mutated in the E1 region.
 4. Thecell of claim 3 wherein the mutation is a deletion.
 5. The cell of claim4, wherein genome of the recombinant adenovirus vector is furtherdeleted for all or part of the E3 region.
 6. The cell of claim 4 whereinthe genome of the recombinant adenovirus vector comprises heterologoussequences.
 7. The cell of claim 6, wherein the heterologous sequencesare inserted at the site formerly occupied by the deleted E1 sequences.8. The cell of claim 6, wherein the heterologous sequences are insertedat the site formerly occupied by the deleted E3 sequences.
 9. The cellof claim 1 wherein the cell is derived from bovine kidney.
 10. The cellof claim 1 wherein the cell is derived from fetal bovine retina.
 11. Thecell of claim 2 comprising adenovirus E1 sequences.
 12. The cell ofclaim 11 wherein the E1 sequences are integrated in the genome of thecell.
 13. The cell of claim 11 wherein the E1 sequences are derived froma human adenovirus.
 14. The cell of claim 13 wherein the humanadenovirus is human adenovirus type 5 (HAd-5).
 15. The cell of claim 14wherein the cell is derived from fetal bovine retina.
 16. The cell ofclaim 15, wherein the genome of the adenovirus vector comprisesheterologous sequences.
 17. The cell of claim 13, wherein therecombinant adenovirus vector is a bovine adenovirus.
 18. The cell ofclaim 16, wherein the recombinant adenovirus vector is a bovineadenovirus.
 19. A cell according to claim 1, wherein the cell comprisesthe genome of a recombinant adenovirus vector, wherein the genome isdeleted for all or part of the adenovirus E1 sequences.
 20. The cell ofclaim 19, wherein the adenovirus genome comprises heterologoussequences.
 21. A method for propagating a recombinant adenovirus genome,the method comprising growth of a recombinant adenovirus vector in acell according to claim
 1. 22. A method for propagating a recombinantadenovirus genome, the method comprising growth of a recombinantadenovirus vector in a cell according to claim
 18. 23. A recombinantadenovirus genome obtained according to the method of claim
 21. 24. Arecombinant adenovirus genome obtained according to the method of claim22.
 25. An immunogenic composition comprising a recombinant adenovirusgenome according to claim
 23. 26. An immunogenic composition comprisinga recombinant adenovirus genome according to claim
 24. 27. A method forpreventing or ameliorating the symptoms of disease, the methodcomprising introduction, into a mammalian subject, of an immunogeniccomposition according to claim
 25. 28. A method for preventing orameliorating the symptoms of disease, the method comprisingintroduction, into a mammalian subject, of an immunogenic compositionaccording to claim
 26. 29. A method for eliciting an immune response ina mammalian host, the method comprising administration of an immunogeniccomposition according to claim
 25. 30. A method for eliciting an immuneresponse in a mammalian host, the method comprising administration of animmunogenic composition according to claim
 26. 31. A method forintroducing a nucleotide sequence of interest into a mammalian cell, themethod comprising contacting the cell with a recombinant adenovirusgenome according to claim
 24. 32. A method for introducing andexpressing a non-adenovirus nucleotide sequence into a mammalian cell,wherein the method comprises contacting the mammalian cell with arecombinant adenovirus vector, wherein the vector comprises arecombinant adenovirus genome according to claim
 24. 33. A cell thatexpresses a function essential for replication of a particular type orspecies of adenovirus, wherein the cell is permissive for thereplication of a recombinant adenovirus vector of a different type orspecies, wherein the recombinant adenovirus vector has a mutation in aregion of its genome corresponding to the essential function provided bythe host cell.
 34. The cell of claim 33, wherein the essentialadenoviral function expressed by the cell is human adenovirus E1function.
 35. The cell of claim 34, wherein the cell is permissive forreplication of a recombinant bovine adenovirus vector having a mutationresulting in the loss of reduction of E1 function.
 36. The cell of claim35, wherein the cell is a bovine cell.
 37. The cell of claim 36, whereinthe cell is derived from fetal bovine retina.